Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca²⁺-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca²⁺-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca²⁺ sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α‐helix. The N‐terminus of Orai proteins contains a conserved CaM‐binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single‐molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N‐termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.     

Influence of stereochemistry and redox potentials on the single- and double-strand DNA cleavage efficiency of Cu(II) and Ni(II) Lys-Gly-His-derived ATCUN metallopeptides

The DNA cleavage chemistry of a series of metallopeptides based on the amino-terminal Cu and Ni (ATCUN) binding motif of proteins has been studied. Specifically, the impact of the positioning of charged Lys side chains and their stereochemistry on metal reduction potentials and DNA cleavage reactivity have been quantitatively evaluated. Both Cu and Ni metallopeptides show a general increase in reactivity toward DNA with an increasing number of Lys residues, while a corresponding decrease in complex reduction potential reflects the enhanced sigma-donor character of the Lys side chain relative to that of Gly. Placement of Lys at the first position in the tripeptide ligand sequence resulted in a greater increase in DNA cleavage reactivity, relative to placement at the second position, while a switch from an l-Lys to a d-Lys typically resulted in enhanced reactivity, as well as perturbations of reduction potential. In the case of Cu peptides, reactivity was enhanced with both increasing positive charge density on the peptide and stabilization of the Cu3+ state. However, for Ni peptides, while the general trends are the same, the correlation with redox behavior was less pronounced. Most likely these differences in specific trends for the Cu and Ni complexes reflect the distinct coordination preferences for Cu3+/2+ and Ni3+/2+ oxidation states, and the consequent distinct positioning of metal-associated reactive oxygen species, as well as the orientation of the DNA-associated complex. Thus, the amino acid composition and stereochemistry of ATCUN metallopeptides can tune the intrinsic reactivities of these systems (their ability to promote formation and activity of metal-associated ROS) as well as their overall structural features, and both of these aspects appear to influence their reactivity and efficiency of DNA strand scission.     

Solution structure of a designed cyclic peptide ligand for nickel and copper ions

Nuclear magnetic resonance (NMR) spectroscopy was used to study a cyclic peptide derived from the amino-terminal copper-and-nickel-binding (ATCUN) motif. The three-dimensional structure of the unliganded peptide in aqueous solution was solved by simulated annealing using distance constraints derived from Nuclear Overhauser Effects. A structural model for the Ni(II)-bound complex was also produced based on NMR evidence and prior spectroscopic data, which are consistent with crystal structures of linear ATCUN complexes. Structural interpolation, or ‘morphing’, was used to understand the transition of this highly structured cyclic peptide from its unliganded structure to its metal-ion-bound structure.     

Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site

Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.     

Incorporation of β-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels,DNA Cleavage and Antiproliferative Activity**

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the β-amino acid β-Ala at position 2 (Gly2→β-Ala2) of the ATCUN motif reinstates ROS production (^•OH and H₂O₂). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β-Ala2 peptide complexes had lower IC₅₀ values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.     

pH-Dependent Cu(II) coordination to amyloid-β peptide: impact of sequence alterations, including the H6R and D7N familial mutations

Copper ions have been proposed to intervene in deleterious processes linked to the development of Alzheimer's disease (AD). As a direct consequence, delineating how Cu(II) can be bound to amyloid-β (Aβ) peptide, the amyloidogenic peptide encountered in AD, is of paramount importance. Two different forms of [Cu(II)(Aβ)] complexes are present near physiological pH, usually noted components I and II, the nature of which is still widely debated in the literature, especially for II. In the present report, the phenomenological pH-dependent study of Cu(II) coordination to Aβ and to ten mutants by EPR, CD, and NMR techniques is described. Although only indirect insights can be obtained from the study of Cu(II) binding to mutated peptides, they reveal very useful for better defining Cu(II) coordination sites in the native Aβ peptide. Four components were identified between pH 6 and 12, namely, components I, II, III and IV, in which the predominant Cu(II) equatorial sites are {-NH(2), CO (Asp1-Ala2), N(im) (His6), N(im) (His13 or His14)}, {-NH(2), N(-) (Asp1-Ala2), CO (Ala2-Glu3), N(im)}, {-NH(2), N(-) (Asp1-Ala2), N(-) (Ala2-Glu3), N(im)} and {-NH(2), N(-) (Asp1-Ala2), N(-) (Ala2-Glu3), N(-) (Glu3-Phe4)}, respectively, in line with classical pH-induced deprotonation of the peptide backbone encountered in Cu(II) peptidic complexes formation. The structure proposed for component II is discussed with respect to another coordination model reported in the literature, that is, {CO (Ala2-Glu3), 3 N(im)}. Cu(II) binding to the H6R-Aβ and D7N-Aβ peptides, where the familial H6R and D7N mutations have been linked to early onset of AD, has also been investigated. In case of the H6R mutation, some different structural features (compared to those encountered in the native [Cu(II)(Aβ)] species) have been evidenced and are anticipated to be important for the aggregating properties of the H6R-Aβ peptide in presence of Cu(II).     

Using nitrile-derivatized amino acids as infrared probes of local environment

It is well-known that the C=N stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, AlaCN and PheCN, in H(2)O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the C=N stretching vibrations of both AlaCN and PheCN shift as much as approximately 10 cm(-1) toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK(579-595)), which contains a single PheCN. MLCK(579-595) binds to CaM in a helical conformation. When the PheCN was substituted on the polar side of the helix, which was partially exposed to water, the C=N stretching vibration is similar to that of PheCN in water. In constrast, when PheCN is introduced at a site that becomes buried in the interior of the protein, the C=N stretch is similar to that of PheCN in THF. Together, these results suggest that the C=N stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies.     

Metal ion selectivity of oligopeptides

Metal binding affinity and selectivity of peptides are reviewed with a special emphasis on the high structural variety of peptide complexes. The most common structural type of these complexes is built up by the deprotonation and metal ion coordination of subsequent amide groups in the form of fused five-membered chelate rings. The metal ion selectivity of this process and the role of various anchoring groups are discussed in detail. The highest metal binding affinity of peptides is connected to the presence of two anchoring groups in appropriate location (the "double anchor"): e.g. the NH2-Xaa-Xaa-His/Cys/Asp/Met-Xaa sequence. Among the side chain donor functions, the imidazole of histidyl and thiolate of cysteinyl residues are the most effective ligating groups and their involvement in metal binding results in a great variety of different macrochelate or loop structures and/or formation of various polynuclear complexes. Examples of these structural motifs and their possible applications have been thoroughly discussed.     

Gas‐phase doubly charged complexes of cyclic peptides with copper in +1, +2 and +3 formal oxidation states: formation,structures and electron capture dissociation

Copper complexes with a cyclic D‐His‐β‐Ala‐L‐His‐L‐Lys and all‐L‐His‐β‐Ala‐His‐Lys peptides were generated by electrospray which were doubly charged ions that had different formal oxidation states of Cu(I), Cu(II) and Cu(III) and different protonation states of the peptide ligands. Electron capture dissociation showed no substantial differences between the D‐His and L‐His complexes. All complexes underwent peptide cross‐ring cleavages upon electron capture. The modes of ring cleavage depended on the formal oxidation state of the Cu ion and peptide protonation. Density functional theory (DFT) calculations, using the B3LYP with an effective core potential at Cu and M06‐2X functionals, identified several precursor ion structures in which the Cu ion was threecoordinated to pentacoordinated by the His and Lys side‐chain groups and the peptide amide or enolimine groups. The electronic structure of the formally Cu(III) complexes pointed to an effective Cu(I) oxidation state with the other charge residing in the peptide ligand. The relative energies of isomeric complexes of the [Cu(c‐HAHK + H)]²⁺ and [Cu(c‐HAHK ? H)]²⁺ type with closed electronic shells followed similar orders when treated by the B3LYP and M06‐2X functionals. Large differences between relative energies calculated by these methods were obtained for open‐shell complexes of the [Cu(c‐HAHK)]²⁺ type. Charge reduction resulted in lowering the coordination numbers for some Cu complexes that depended on the singlet or triplet spin state being formed. For [Cu(c‐HAHK ? H)]²⁺ complexes, solution H/D exchange involved only the N–H protons, resulting in the exchange of up to seven protons, as established by ultra‐high mass resolution measurements. Contrasting the experiments, DFT calculations found the lowest energy structures for the gas‐phase ions that were deprotonated at the peptide C_α positions. Copyright © 2012 John Wiley & Sons, Ltd.     

The role of the βAsp residue in copper(II) binding by modified peptides

The synthesis and binding abilities of peptides containing β-amino acids towards Cu(II) ions are presented. The peptides studied were: Ala-βAsp-Ser-Gly and Arg-Lys-βAsp-Val-Tyr. Potentiometric titrations were carried out to establish the stoichiometry of the resulting metal-ligand complexes. The copper(II) coordination mode of the complexes was investigated by performing detailed spectroscopic analyses (UV–Vis, CD) in strict correlation with potentiometric measurements. The results obtained on the β-peptides studied allowed the characterization of the influence of this structural modification on the coordination abilities of the peptides. Moreover, the role of the α-Asp position in the peptide chain was also described.     

Molecular modeling of class I and II alleles of the major histocompatibility complex in <Emphasis Type="Italic">Salmo salar</Emphasis>

Knowledge of the 3D structure of the binding groove of major histocompatibility (MHC) molecules, which play a central role in the immune response, is crucial to shed light into the details of peptide recognition and polymorphism. This work reports molecular modeling studies aimed at providing 3D models for two class I and two class II MHC alleles from Salmo salar (Sasa), as the lack of experimental structures of fish MHC molecules represents a serious limitation to understand the specific preferences for peptide binding. The reliability of the structural models built up using bioinformatic tools was explored by means of molecular dynamics simulations of their complexes with representative peptides, and the energetics of the MHC-peptide interaction was determined by combining molecular mechanics interaction energies and implicit continuum solvation calculations. The structural models revealed the occurrence of notable differences in the nature of residues at specific positions in the binding groove not only between human and Sasa MHC proteins, but also between different Sasa alleles. Those differences lead to distinct trends in the structural features that mediate the binding of peptides to both class I and II MHC molecules, which are qualitatively reflected in the relative binding affinities. Overall, the structural models presented here are a valuable starting point to explore the interactions between MHC receptors and pathogen-specific interactions and to design vaccines against viral pathogens.     

Direct Observation of Ca2+‐Induced Calmodulin Conformational Transitions in Intact Xenopus laevis Oocytes by 19F NMR Spectroscopy

The Ca²⁺‐mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by ¹⁹F NMR spectroscopy of the transition of the Ca²⁺‐free and ‐bound forms in Xenopus laevis oocytes at different Ca²⁺ levels. We find that the Ca²⁺‐bound CaM population increased greatly upon binding the target protein myosin light‐chain kinase (MLCK) at the same Ca²⁺ level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long‐range structural constraints in cells. Our study shows that ¹⁹F NMR spectroscopy can be used to obtain long‐range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants.     

Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates

A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/hydroxyl radical) = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.     

多肽与花粉钙调素的相互作用及对细胞功能的影响

研究了在Ca²⁺存在下,丹磺酰标记的花粉钙调素(D-pCaM)与合成的多肽相互作用的规律.研究结果表明,被研究的绝大多数多肽能与D-pCaM结合而形成复合物,使D-pCaM的荧光光谱发生变化.用荧光法测定了这些复合物的解离常数K_d,其中肽BP-13的Kd值为4.0nmol/L,是与pCaM结合能力最强的一个多肽.除证明了多肽的疏水性,预测二级结构形成倾向和链长对其与pCaM结合能力有影响外,还发现用D-丙氨酸取代肽链中的Gly或L-丙氨酸后,可改变肽对pCaM的亲和性,这为提高多肽与钙调素的亲和力提供了一条改进的途径.我们的研究还可以定性地说明不同来源CaM性质相似又有差异,反映了它们的高度保守性和变异性.同时还展示了所研究的多肽对细胞信息传导过程的影响,为研究CaM对细胞功能调节的作用机制和建立pCaM拮抗肽的活性筛选模型提供了思路.     

Ranking Peptide Binders by Affinity with AlphaFold**

AlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein-peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.     

Mass Spectral Studies Reveal the Structure of Aβ1-16-Cu(2+) Complex Resembling ATCUN Motif

In Alzheimer's disease, copper binds to amyloid beta (Aβ) peptide and generates oxidative stress. The coordination of histidine (His) residues to Cu(2+) is still uncertain. We studied Cu(2+) binding to Aβ1-16 peptide using the diethyl pyrocarbonate (DEPC) assay and mass spectrometry. Our results show that only one His is involved in Cu(2+) coordination, which is identified as His6 using mass spectral studies. Novel nickel displacement studies have further supported the proposal that the Cu(2+) binding site of Aβ1-16 peptide resembles the ATCUN motif of human serum albumin.     

Model peptides based on the binding loop of the copper metallochaperone Atx1: selectivity of the consensus sequence MxCxxC for metal ions Hg(II), Cu(I), Cd(II), Pb(II), and Zn(II)

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intracellular concentration of ions such as Cu(I), Hg(II), Zn(II), Cd(II), and Pb(II). A relevant task is thus the selectivity of the motif MxCxxC for these different metal ions. To analyze the coordination properties and the selectivity of this consensus sequence, we have designed two model peptides that mimic the binding loop of the copper chaperone Atx1: the cyclic peptide P(C) c(GMTCSGCSRP) and its linear analogue P(L) (Ac-MTCSGCSRPG-NH2). By using complementary analytical and spectroscopic methods, we have demonstrated that 1:1 complexes are obtained with Cu(I) and Hg(II), whereas 1:1 and 1:2 (M:P) species are successively formed with Zn(II), Cd(II), and Pb(II). The complexation properties of the cyclic and linear peptides are very close, but the cyclic compound provides systematically higher affinity constants than its unstructured analogue. The introduction of a xPGx motif that forms a type II beta turn in P(C) induces a preorganization of the binding loop of the peptide that enhances the stabilities of the complexes (up to 2 orders of magnitude difference for the Hg complexes). The affinity constants were measured in the absence of any reducing agent that would compete with the peptides and range in the order Hg(II) > Cu(I) > Cd(II) > Pb(II) > Zn(II). This sequence is thus highly selective for Cu(I) compared to the essential ion Zn(II) that could compete in vivo or compared to the toxic ions Cd(II) and Pb(II). Only Hg(II) may be an efficient competitor of Cu(I) for binding to the MxCxxC motif in metalloproteins.     

Gas-phase noncovalent interactions between vancomycin-group antibiotics and bacterial cell-wall precursor peptides probed by hydrogen/deuterium exchange

Gas-phase structures of noncovalent complexes between the glycopeptide antibiotics vancomycin, eremomycin, ristocetin, and pseudo aglyco-ristocetin and the cell-wall mimicking peptides N-acetyl-D-Alanyl-D-Alanine, N-acetyl-Glycyl-D-Alanine, and N,N′-di-acetyl L-Lysyl-D-Alanyl-D-Alanine have been probed by hydrogen/deuterium (H/D) exchange using ND₃ as reagent gas. The noncovalent complexes were transferred from solution to the vacuum using electrospray ionization. The H/D exchange of the solvent-free ions was studied in a Fourier transform ion cyclotron resonance mass spectrometer. The H/D exchange behavior of the free antibiotics and the free peptides were compared with the exchange observed for the antibiotic–peptide complexes. A general increase was found in the degree of deuterium incorporation upon complex formation with the ligand, which indicates that the peptide binding makes more sites on the antibiotic capable of taking part in the H/D exchange. Apart from H/D exchange, adduct formation with ND₃ was observed, but only for the singly protonated peptides and the doubly protonated [ristocetin+N-acetyl-D-Alanyl-D-Alanine]. This marked difference in chemical reactivity of closely related systems such as [ristocetin+N-acetyl-Glycyl-D-Alanine] and [ristocetin+N-acetyl-D-Alanyl-D-Alanine] indicates that the gas-phase structures of these noncovalent complexes are quite sensitive to small changes in the primary structures of the peptides. The gas-phase structures of the antibiotic–peptide complexes are probably different from the solution-phase structures, with the peptides no longer bound to the characteristic solution-phase binding pockets of the antibiotics.     

Mathematical modeling of the myosin light chain kinase activation

The mathematical model presented here describes the interactions among Ca2+, calmodulin (CaM), and myosin light chain kinase (MLCK) and consists of a kinetic scheme taking into account 7 reactions instead of 12 as proposed previously. We derive a system of 5 nonlinear ordinary differential equations. Solving it yields the prediction of active MLCK as a function of [Ca2+] whereby the active MLCK is defined to be proportional to the Ca4CaM.MLCK complex concentration. The model predictions are compared with other theoretical and experimental predictions of active MLCK as well as with the results of our previously proposed complex model.